1. Field of the Invention
The presently claimed and disclosed inventive concept(s) relates, in general, to heparosan analogs, as well as the synthases responsible for production of said heparosan analogs. The presently disclosed and claimed inventive concept(s) is also related to methods of production and use of the heparosan analogs and the heparosan analog synthases.
2. Brief Description of the Related Art
Certain pathogenic microbes employ extracellular capsules of host-like glycans to evade host defenses and to increase virulence. Previous work by the inventors and others has identified very distinct types of microbial glycosaminoglycan [GAG] synthases, the bifunctional enzymes that assemble GAG polysaccharides. These synthases include peripheral membrane-associated two domain enzymes such as the Pasteurella multocida GAG synthases PmHAS (hyaluronan) (DeAngelis et al., 1996 and 1998), PmCS (chondroitin) (DeAngelis et al., 2000), PmHS1 and PmHS2 (heparosan) (DeAngelis et al., 2002 and 2004) (FIG. 1) and KfoC (chondroitin) from Escherichia coli K4 (Ninomiya et al., 2002), as well as integral membrane proteins with unknown domain structures such as the Streptococcus pyogenes hyaluronan synthase SpHAS (DeAngelis et al., 1993) and the Chlorella virus PBCV-1 hyaluronan synthase CvHAS (DeAngelis et al., 1997). All of the known GAG synthases employ UDP-sugar precursors to form the repeating disaccharide units (DeAngelis, 2002). The Streptococcus hyaluronan synthase has some similarity to vertebrate hyaluronan synthases at the amino acid sequence level (Weigel et al., 2007), but the bacterial chondroitin and heparosan synthases are quite different to their vertebrate counterparts (DeAngelis et al., 2000 and 2002).
Comomonas testosteroni (Ct) is a Gram-negative aerobic bacteria that is found in diverse environments (Ma et al., 2009). Bacteria of the genus Comamonas are predominant in activated sewage sludge (Dias et al., 1964) and are defined by a poor ability to use carbohydrates; instead carbon is derived from molecules such as testosterone and other cyclic hydrocarbons (Horinouchi et al., 2010; Linares et al., 2008). C. testosteroni has recently been identified as an opportunistic human pathogen that has been found in various hospital infections including meningitis (Arda et al., 2003; Jin et al., 2008), bacteremia (Gul et al., 2007), and endophthalmitis (Reddy et al., 2009). The ability for C. testosteroni to survive and thrive in such diverse environments, as well as its potential use for cleaning up environmental contamination with xenobiotic compounds such as polychlorinated biphenyls and linear alkylbenzenesulfonate make it a particularly interesting organism (Schleheck et al., 2004 and 2010). There is only one published study indicating the presence of a mucoid exopolysaccharide capsule of C. testosteroni A20 (Bossier et al., 1996); however, there is no genomic information available for this strain, and the nature of the polysaccharide was not determined.
Heparin is a useful drug widely used in hospitals as an anticoagulant. Heparin also has other potential uses in combating diseases such as cancer and inflammation, but the polysaccharide's anticoagulant effects make it difficult to use for these other promising indications (i.e., patients may have excessive bleeding). Also, heparin is currently derived from animal products (e.g., porcine intestinal mucosa, bovine lung); thus, its method of preparation must remove adventitious agents, antigens, etc., and its potency is variable. In addition, due to collection of almost all pigs slaughtered around the world for food, the supply chain is not secure, as evidenced by the contaminated heparin from China in 2008 that resulted in many deaths. Therefore, microbial-derived heparin substitutes are desirable; however, the manufacture using heparosan (unsulfated, unepimerized backbone of heparin) as the precursor for heparin requires many steps to convert into heparin or heparin-like material.
Thus, there is a need in the art for new non-animal-derived heparosan analogs that may be simpler to convert into anticoagulants or other therapeutics.